Name: colonization of euprymna scolopes squid by vibrio fischeri
Uploaded: 2012-03-01T13:00:00
Description: Watch this Scientific Journal Video about Colonization of Euprymna scolopes Squid by Vibrio fischeri at JoVE.com

Forfatterne takker Mattias Gyllborg efter blæksprutter facilitet støtte og for kommentarer til dette manuskript, Michael Hadfield og Kewalo Marine Laboratory for hjælp under feltet indsamlingen, og medlemmer af Ruby og McFall-Ngai Laboratorium for bidrag til denne protokol. Arbejdet i Mandel Laboratory understøttes af NSF IOS-0.843.633.

1. Fremstillinq af bakteriel Podestoffer <ol><li> Dag 0 To dage før blæksprutte podningen, plade de relevante bakteriestammer på LBS 13 agar. </li><li> Inkuber bakterier ved 25-28 ° C natten over. </li><li> Dag 1 Inokulere 3 ml LBS medium i et glas kultur rør med en koloni af hver V. fischeri stamme for infektion. Forbered dobbelt rør som backup. </li><li> Dag 2 (Koordiner bakterielle trin 1,4-1,6 med Squid trin 3...

<ol>
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Den kolonisering beskrevne analyse giver mulighed for analyse af en naturlig symbiotisk proces i et kontrolleret laboratorium omgivelser. Som sådan kan den anvendes til at vurdere kolonisering af mutante stammer af forskellige naturlige isolater, og under forskellige kemiske systemer. Variationer i de beskrevne eksperimenter er almindeligt anvendt til at vurdere forskellige aspekter af symbiose. Kinetikken for kolonisering kan måles ved at undersøge luminescens under de første 24 timer, hvilket kan detekteres automa...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalognr </td><td> Kommentarer </td></tr><tr><td> Glas Kultur Rør, 16 mm i diameter </td><td> VWR </td><td> 47729-580 </td><td></td></tr><tr><td> Caps til Glas Kultur Tubes </td><td> Fisher </td><td> NC9807998 </td><td></td></tr><tr><td> Synlig Spektrofotometer til bestemmelse af OD 600 </td><td> Biowave </td><td> CO8000 </td><td> Enhver spektrofotometer kan måle OD 600 vil arbejde. Denne enhed kan måle OD600 af væske direkte i glasset dyrkningsrør. Vis justering af podestoffet beregningen kan være nødvendigt, afhængigt af det anvendte instrument. </td></tr><tr><td> GloMax 20/20 Single-Tube Luminometer </td><td> Promega </td><td> E5311 </td><td> Svarende til Turner BioSystems 20/20n Luminometer. Omfatter mikrocentrifugerør holderen. </td></tr><tr><td> GloMax 20/20 Lys Standard </td><td> Promega </td><td> E5341 </td><td> For luminometer kalibrering. </td></tr><tr><td> Refraktometer, Handheld </td><td> Foster og Smith Aquatics </td><td> CD-14.035 </td><td> Kalibrere før hver anvendelse med deioniseret vand. Skylles efter hver anvendelse med deioniseret vand for at undgå salt opbygning. </td></tr><tr><td> Instant Ocean (kunstigt havvand koncentrat) </td><td> Foster &amp; Smith Aquatics </td><td> CD-16.881 </td><td> Forberede ved 35 ‰ i deioniseret vand under anvendelse af et refraktometer, filtreres derefter gennem et 0,2 um SFCA filter. </td></tr><tr><td> Filtreringsenhed </td><td> Nalgene </td><td> 158-0020 </td><td> Uden overfladeaktivt middel celluloseacetat (SFCA) membran, 0,2 um. Vi har observeret forskellige resultater med nogle overfladeaktivt indeholdende PES filtre. </td></tr><tr><td> Overførselspipetter </td><td> Fisher </td><td> 13-711 -09:00 </td><td> Ved hjælp af en saks eller barberblad, skære spidsen rent over den første kant til at øge diameteren af ​​pipettespidsen og undgå klemning blæksprutte larver. </td></tr><tr><td> Engangs Prøve Skåle (plast tumblers) </td><td> Comet </td><td> T9S (9 oz.) </td><td> Skåle til inokulering med øvre diameter 3 ¼ &quot;lavere diameter på 2 ¼&quot;, højde 3 &quot;. Skåle skabe et homogent miljø, da de ikke har nogen bundrand, hvor blæksprutte kan blive fanget i en lav-oxygen niche. Størrelsen optimeres for 40-ml inokulum. Findes på webstaurantstore.com, # 619PI9. </td></tr><tr><td> Drosophila Hætteglas </td><td> VWR </td><td> 89092-720 </td><td> Vial diameter passer til åbningen på luminometeret PMT. </td></tr><tr><td> 1,5 ml mikrocentrifugerør </td><td> ISC Bioexpress </td><td> C-3217-1CS </td><td> Rør skal passe til formen på pestles. </td></tr><tr><td> Ethanol, 200 Proof </td><td> Fisher </td><td> BP2818-100 </td><td></td></tr><tr><td> Pestles </td><td> Kimble Chase / Kontes </td><td> 749521-1500 </td><td></td></tr><tr><td> Belægning perler, 5 mm i diameter </td><td> Kimble Chase </td><td> 13500 5 </td><td> Fremstilling af 5 pr rør og autoklaven. </td></tr></tbody></table>

colonization of euprymna scolopes squid by vibrio fischeri

Specifikke bakterier findes i association med dyrevæv 1-5. Sådanne værts-bakterielle foreninger (symbioser) kan være skadelig (patogene), har ingen egnethed konsekvens (kommensal), eller være en fordel (mutualistisk). Mens megen opmærksomhed er blevet givet til sygdomsfremkaldende interaktioner, vides kun lidt om de processer, der dikterer den reproducerbare købet af gavnlige / kommensale bakterier fra omgivelserne. Den lys-organ mutualism mellem marine Gram-negativ bakterie V. fischeri og Hawaiian stumphale blæksprutte, E. scolopes, repræsenterer en meget specifik interaktion, hvor en vært (E. scolopes) etablerer et symbiotisk forhold med kun en bakteriearter (V. fischeri) under hele løbet af sin levetid 6,7. Bioluminescens er fremstillet af V. fischeri i denne interaktion tilvejebringer en anti-eliminerende fordel E. scolopes under natlige aktiviteter 8,9, mensdet næringsrige værtsvævet giver V. fischeri med en beskyttet niche 10. Under hver vært generation, er dette forhold gentaget, hvilket repræsenterer en forudsigelig proces, der kan vurderes i detaljer på forskellige stadier af symbiotisk udvikling. I laboratoriet, hvis opsamlet inden for de første 30-60 min og overførtes til symbiont-frit vand juvenile blæksprutte lugen aposymbiotically (uncolonized), og kan ikke blive koloniseret undtagen ved den eksperimentelle inokulum 6. Denne interaktion giver således en nyttig model system til at vurdere de enkelte trin, der fører til konkrete køb af en symbiotisk mikrobe fra miljøet 11,12. Her beskriver vi en metode til at vurdere graden af kolonisering der opstår, når nyklækkede aposymbiotic E. scolopes udsættes for (kunstige) havvand indeholdende V. fischeri. Denne enkle analyse beskriver podning, naturlig infektion, og inddrivelseaf den bakterielle symbionten fra den spirende lys organ E. scolopes. Care er taget til at give en konsistent miljø for dyrene under symbiotisk udvikling, især med hensyn til vandkvalitet og lette signaler. Metoder til at karakterisere det symbiotiske beskrevne population omfatter (1) måling af bakterielt afledte bioluminescens, og (2) direkte koloni optælling af genvundne symbionter.

Watch this Scientific Journal Video about Colonization of Euprymna scolopes Squid by Vibrio fischeri at JoVE.com

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Metoden beskriver den procedure, som Hawaiian stumphale blæksprutte,<em&gt; Euprymna scolopes</em&gt; Og bakterielle symbiont,<em&gt; Vibrio fischeri</em&gt; Er hævet separat og indføres derefter for at muliggøre specifik kolonisering af blæksprutte lys organ af bakterierne. Kolonisering påvisning af bakterielt afledte luminescens og ved direkte kolonitælling er beskrevet.

Kolonisering af Euprymna scolopes Squid ved Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

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Colonization of Euprymna scolopes Squid by Vibrio fischeri

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